1. Field of the Present Invention
This invention relates to a method for injection and stacking of analytes in high salt samples. This stacking method works with both pressure injection or electrokinetic injection of high salt samples. This electrokinetic injection method allows for translation of high-salt stacking from the capillary to the microchip format.
2. Description of the Prior Art
Capillary electrophoresis (CE) is used for the analysis of a wide variety of compounds according to size and charge. CE is beneficial because it allows for high resolution separations while using miniscule amounts of both samples and reagents. However, neutral compounds cannot be separated in this mode because they have no charge. Therefore, micellar capillary electrophoresis (MCE) was pioneered to separate neutral and charged species.
MCE uses micellar solutions to separate neutral and charged species. The addition of a charged micelle solution in the separation buffer allows for hydrophobic interaction between neutral analytes and the electrophorectically-mobile micelle phase which can afford separation. However, in MCE (as in all CE modes), the total capillary volume is typically in the microliter range. Therefore injection lengths are limited by the micrometer dimensions of the capillary to the millimeter range. As a result it is critical to develop sample stacking schemes which allow for longer injections without a loss of resolution of analytes.
However, sample stacking in MCE oftentimes proves problematic as most known stacking methods require a particular combination of micelle type, sample pH, electroosmotic flow rate, and separation-mode polarity. Further, the difference in ionic strength between separation buffer and sample matrix is oftentimes achieved by diluting the sample, which compromises resolution. The need exists for a simple, more universal stacking method not limited to micelle type, sample pH, electroosmotic flow rate, and separation-mode polarity. The need also exists for a stacking method useful for neutral species.
The present invention is directed to a method of preparing a device for electrophoresis which includes the step of achieving a desired conductivity in a sample of analytes through any appropriate mechanism. Such mechanisms can include the addition of salts, the addition of conductive species not considered to be salts, the addition of pH buffers, the addition of conductive species having a higher electrophoretic mobility than that of the buffer ions, the elimination of organic modifiers (which decreases conductivity), the utilization of naturally high-conductivity samples and the dilution of high-conductivity by the addition of organic modifiers or water. The conductivity of the sample analytes is measured relative to a buffer, in which electrophoretic separation occurs. The desired conductivity of the sample of analytes may be achieved by modifying the characteristics of the buffer through any appropriate mechanism. Such mechanisms can include the reduction or elimination of salts, the reduction or elimination of conductive species not considered to be salts, the reduction or elimination of pH buffers, the maintenance, addition, or elimination of conductive species with electrophoretic mobility which are lower than the mobility of the sample matrix conductive species, the addition of organic modifiers, or the addition of conductive species.
A plug of the sample is injected either through pressure or by applying an electric field into a capillary (a filament type of device), a microchannel on a microchip, or any other type of device in which electrophoretic separation can occur.
An electric field is applied which causes field amplification in the separation buffer. That causes stacking of buffer ions against the sample which subsequently causes stacking of the sample components. Stacking of the sample components is believed to cause reverse isotachophoresis due to the higher mobility anion in the sample matrix occurring at a higher concentration than the separation buffer ion. That causes stacking of the separation buffer ion against the sample which subsequently causes stacking of the sample components. When the sample is electrokinetically injected, the foregoing processes begin during electrokinetic injection and continue after the electrokinetic injection is completed.
One benefit of the present invention is that it provides a method of stacking that is independent of the pH, micelle type, electroosmotic flow, and separation-mode polarity. This method is also applicable to a wide variety of buffer systems and does not require sample dilution.
Electrokinetic injection is counterintuitive because a neutral analyte will have no electrophoretic mobility in an electric field in the absence of a carrier vector such as a micelle. Therefore electroinjection of neutral analytes without a carrier vector is a novel benefit of the present invention. This method of the present invention, that is, utilizing electroinjection, is potentially more than an order of magnitude more powerful for concentrating neutral analytes than prior art mechanisms.
Additionally, as the present invention can utilize electrokinetic injection, it allows translation of high-salt stacking from the capillary to the microchip format. The method of the present invention can be translated directly to the microchip format with existing channel technology, that is, a simple separation channel with a cross-T injection channel. It is possible to utilize the microchip format because the present invention may be driven electrokinetically, which is currently the only practical mode of sample injection on the microchip format. Those, and other advantage and benefits of the present invention will become apparent from the Detailed Description of the Preferred Embodiment herein below.